Process for Production of Diphtheria Toxin

ABSTRACT

The present invention provides a method of purifying diphtheria toxin comprising (1) fermenting a microorganism strain capable of producing diphtheria toxin using glucose as a carbon source, said method comprising adding glucose to a growing culture whereby the addition of glucose maintains microorganism growth effective to support diphtheria toxin production; and (2) purifying the diphtheria toxin from the culture by contacting a toxin containing preparation derived therefrom with an ion exchange matrix, eluting a fraction containing the toxin, applying the eluate to a hydrophobic matrix, and eluting a fraction containing the toxin.

This application is a divisional filing of application Ser. No.11/204,193 filed Aug. 15, 2005, now U.S. Pat. No. 7,273,728, grantedSep. 25, 2007, which is a divisional filing of application Ser. No.10/690,855 filed Oct. 22, 2003, now U.S. Pat. No. 6,962,803, grantedNov. 8, 2005, which is a divisional of application Ser. No. 09/914,162filed Jan. 25, 2002, now U.S. Pat. No. 6,689,871 granted Feb. 10, 2004which is a 35 U.S.C. § 371 filing of international application numberPCT/GB00/00680 filed Feb. 25, 2000 which claims priority to applicationnumber 9904582.5 filed in Great Britain on Feb. 26, 1999.

The present invention relates to a process for producing mutant forms ofdiphtheria toxin, and in particular to a process for producing anon-toxic mutant of diphtheria toxin, for example the mutant known asCRM107, and a toxic conjugate thereof, which can be used for therapeuticpurposes.

Diphtheria toxin is a proteinaceous toxin which is synthesised andsecreted by toxigenic strains of Corynebacterium diphtheriae, i.e.strains which are lysogenic for a bacteriophage carrying the toxin gene.It is initially synthesised as a 535 amino acid polypeptide whichundergoes proteolysis to form the toxin which is composed of twosubunits, named A and B, joined by a disulphide bond. The A subunit isthe enzymatic domain. It catalyses the ADP ribosylation of ElongationFactor 2, thereby inactivating EF-2. EF-2 is an essential enzymeinvolved in protein synthesis, and its inactivation results in cessationof protein synthesis and death of an ‘infected’ eucaryotic cell. The Asubunit is only active intracellularly, but since alone it is unable tobind to or cross the cell membrane it is not toxic when appliedextracellularly. It is the B subunit which is responsible for gettingthe active A subunit into the cells; it does this by binding to thesurface of cells by means of a cell surface receptor and then itfacilitates the passage of the A subunit across the cell membrane intothe cytoplasm where the toxic effects of the A subunit may be exerted.

Diphtheria toxin is highly cytotoxic; a single molecule can be lethalfor an ‘infected cell’ and a dose as low as 10 ng/kg can kill animalsand humans. There has thus been some considerable interest ininvestigating therapeutic strategies which utilise the toxic A subunit.The native toxin whilst being highly cytotoxic is non-specific, i.e. itwill attack any cell which carries a receptor for the B subunit.

Certain mutant forms of the diphtheria toxin have been reported whichare deficient in the cell binding and/or translocation function. Theseinclude toxin molecules which have a mutation in the B subunit whichresults in reduced binding to cells, such as for example mutants CRM9,CRM 45, CRM102, CRM103 and CRM107, as described by Nicholls & Youle inGenetically Engineered Toxins, Ed: Frankel, Marcel Dekker, Inc, 1992.The resulting toxin molecules are essentially non-toxic since the Asubunit is unable to reach its site of action. These mutations can havea dramatic effect. Thus CRM107 has an amino acid substitution atposition 525, where serine in the native toxin has been replaced byphenylalanine, resulting in a more than 1000 fold reduction in the cellbinding property with little or no effect on the translocatingproperties of the B subunit. The A subunit in such mutants isunaffected, and, if it can be targeted into the cytoplasm, is as toxicas the native toxin.

In designing cytotoxic drugs, there is thus interest in utilising thesemutant forms of diphtheria toxin to target specific cell populationswithout affecting normal cells, by modifying the mutant toxin by linkingit in some way to a moiety which is capable of binding to cells, and inparticular to a moiety which is specific for certain cells or celltypes, such as an antibody to a specific receptor, or a moiety such as aprotein for example transferrin, which has a binding partner e.g. in theform of a receptor expressed only or at least predominantly on thesurface of cells which are to be killed. In this way, it is possible toharness the cytotoxic properties of the diphtheria toxin A subunit,without affecting, or with only limited effect on, normal cells.

One area where modified forms of mutant diphtheria toxin such asmodified CRM107 may be used is in the treatment of certain cancerousconditions, and in particular malignant gliomas. Malignant glioma is themost common CNS neoplasm in adults. No therapy is currently availableand prognosis of patients with high grade gliomas, anaplasticastrocytomas and glioblastoma multiforme is thus bleak, with deathusually occurring within one year of diagnosis. Mutated diphtheria toxinCRM107, particularly in the form of a targeted conjugate, provides atherapy for conditions such as this, and in particular, conjugates ofCRM107 with the iron binding protein transferrin. This is particularlysuitable for treatment of tumours including brain neoplasms becausetransferrin receptors are expressed at a high level on the surface ofrapidly dividing cells such as glioma cells, but are absent on thesurface of normal brain tissue. Thus mutated diphtheriatoxin-transferrin conjugates may be selectively targeted to neoplastictissue, where the toxin is internalised, and the A subunit kills the‘infected’ cell.

For clinical use, large quantities of mutant diphtheria toxin areneeded. There are however problems in producing diphtheria toxin fromtoxin producing strains of C. diphtheriae, and moreover, difficultieshave been encountered in scaling up laboratory scale fermentationconditions to produce sufficient quantities of toxin, and in particularmutant forms of diphtheria toxin, for therapeutic use. Thus there areproblems in obtaining toxin in sufficient yield and purity and largescale production thus tends to be inefficient. These difficulties needto be overcome in order to be able to exploit the promise of theseso-called targeted mutant toxin derived drugs.

It is known in the art that diphtheria toxin production is dependent onthe conditions under which the producing strain is grown. In particular,both iron content of the growth medium and the carbon source which areessential for bacterial growth have been found to have an effect ontoxin production. Thus, it has been known for some time that iron inlarge concentrations has an inhibitory effect upon toxin production, inother words, toxin production is negatively regulated by iron. Thus fortoxin preparation, low iron growth media is used, with iron generally inthe range of 50-100 μg/l.

Whilst glucose is commonly used as a carbon source for bacterial growth,it has also been known for some time that fermentation of glucose by C.diphtheriae can affect diphtheria toxin production. Thus it is knownthat fermentation of glucose by C. diphtheriae can lead to acidicfermentation products including acetic, formic and lactic acid, at leastsome of which are thought to be bacteriostatic and even possiblybactericidal for the bacteria. Glucose fermentation thus can affect therate of bacterial growth with corresponding effects on toxin production.It has also been proposed that the acidic fermentation products may havean effect on the stability of the toxin. For this reason, other carbonsources such as maltose and glycerol, used either as the sole carbonsource, or as an at least partial substitute for glucose have been usedin the art for culturing toxin producing strains of C. diphtheriae.Neither of these carbon sources is however as efficient an energy sourceas glucose.

We have now developed a new fermentation process which enablesdiphtheria toxin to be produced by C. diphtheriae in good yieldutilising glucose as the carbon source.

Thus viewed from one aspect, the present invention provides a method forthe production of diphtheria toxin wherein a microorganism capable ofproducing diphtheria toxin is fermented using glucose as a carbonsource, said method comprising adding glucose to a growing culturewhereby the addition of glucose maintains a microorganism growtheffective to support diphtheria toxin production.

As used herein, the term ‘diphtheria toxin’ is used to refer to thenaturally occurring protein, as well as mutated forms, particularly fortherapeutic purposes mutated forms of the B subunit which have reducedor no binding function whilst retaining at least a degree oftranslocation function and preferably retaining at least some A subunitenzymatic activity, variants for example proteins which have amino acidsubstitutions, additions or deletions and fragments thereof,particularly fragments which retain the cytotoxic activity of the Asubunit. The method of the invention may be used to prepare thenaturally occurring diphtheria toxin, as well as mutant forms, such asthe aforementioned non-toxic mutants such as CRM107.

The naturally occurring diphtheria toxin may be obtained from toxinproducing strains available from a variety of publicly available sourcesincluding the American Type Culture Collection. CRM 107 may be obtainedfrom the strain Corynebacterium diphtheriae monolysogen C7 β^(tox 107),which is obtainable from National Institutes of Health, 6011 ExecutiveBoulevard, Rockville Md. 20852, USA (Dr Richard Youle). Mutant forms ofthe toxin, such as the mutant forms described by Laird et al, J.Virology, 19, 220-227, 1976, and by Nicholls & Youle in GeneticallyEngineered Toxins, Ed: Frankel, Marcel Dekker, Inc, 1992, includingCRM107, may also be prepared by methods known in the art, for example bythe methods described in Laird et al (supra) or by expression in C.diphtheriae or other microorganisms using the techniques of recombinantDNA technology (Sambrook et al, Molecular Cloning, a Laboratory Manual,Cold Spring Harbor Laboratory Press, 1989), and also by site directedmutagenesis, based on the known nucleotide sequence (Greenfield et al,Proc Nat Acad Sci 50, 6953-7, 1993) of the wild type structural gene fordiphtheria toxin carried by corynebacteriophage β.

In the method of the invention, glucose is added as required to agrowing culture, preferably an exponential culture, more preferably alate exponential phase culture, and/or may be added as required to agrowing culture in stationary phase, preferably a batch fermentationculture.

This method contrasts with conventional batch fermentation, wherein aninitial supply of nutrients is not renewed, and thus the culture growsexponentially for only a few generations until an essential nutrient isexhausted, and with conventional continuous culture in which freshgrowth medium is added continuously at a constant rate whilst culture issimultaneously removed resulting in much longer exponential growthperiods.

The fed batch method according to the invention offers an advantage overprior methods which utilise glucose as a carbon source for growth of C.diphtheriae and synthesis of toxin. Thus for example, Fass et al., Appl.Microbiol. Biotechnol. 43, 83-88 (1995) describe a two stage process inwhich glucose and iron are simultaneously depleted at the end of theexponential growth phase, and toxin is thus produced only once theculture enters stationary phase. In contrast, the controlled glucoseaddition in accordance with the invention enables toxin to be producedduring the late exponential phase as well as during the stationary phaseby prolonging the late exponential and early stationary phases, and as aresult enables toxin to be produced and accumulated for a longer timethereby increasing the cumulative toxin production capacity of aculture.

The method may be used to produce diphtheria toxin both from its naturalproducer, C. diphtheriae and also from other microbial hosts, such asbacteria for example E. coli transformed with toxin genes or modifiedtoxin genes.

The fed batch fermentation method is particularly applicable for theproduction of diphtheria toxin by toxin producing strains of C.diphtheriae because of the inhibitory effect which certain nutrients ortheir metabolites may have on toxin production, if their concentrationis not controlled carefully. The fed batch method of the invention thusenables a low level of glucose to be maintained in the medium whilst atthe same time providing the necessary carbon source required for growth.Thus by carefully monitoring the levels of glucose and/or pH of thefermentation culture, glucose levels may be controlled to achieve abalance between on the one hand providing sufficient and efficientcarbon source to support bacterial growth, and toxin production, and onthe other hand limiting the generation of inhibitory fermentationby-products which can have a detrimental effect on toxin production. Byadding glucose to the fermentation medium as required during thefermentation, the pH can be controlled using the organic acids producedfrom glucose metabolism without the additional complication of addingexogenous inorganic acid, whilst enabling toxin to be produced duringboth the exponential and stationary growth phases. Thus glucose can beused both as a source of acid and as a carbon source to increase celldensity.

In the method of the invention, growth is initiated in a glucose-basedmedium comprising conventional levels of glucose, for example within therange 0.8 to 2.5% such as about 1.5% glucose. The improvement over theprior methods is that glucose is added to the culture medium during thefermentation, commencing during the exponential growth phase or duringthe stationary phase at such time as is required to maintain the pHwithin a range optimal for microorganism growth and toxin production,preferably from 7.0 to 7.5, preferably from 7.1 to 7.3, for example ataround 7.2. As mentioned above, certain acidic substances are byproducts of glucose metabolism, and these acids assist in controllingthe pH of the fermentation medium.

If necessary, the pH may additionally be controlled by addition of acidor base as appropriate to maintain the pH of the culture at anappropriate level for toxin expression, for example at about pH 7.2±0.2.

Thus we have found that by controlling the glucose concentration withinthe fermentation culture by periodic addition to maintain the pH withinthe aforementioned range, it is possible to maximise cell growth andtoxin production by minimising toxin degradation due to fluxes in pH.Thus we have observed that as the C. diphtheriae cells enter thelogarithmic growth phase, pH and glucose concentration decreases. Wehave also found that the purity of the toxin decreased as the pH of themedium decreased. Without wishing to be bound by theory, it is believedthat this might be due to proteolytic breakdown. Toxin purity was alsoobserved to decrease as fermentation progressed at pH's below about 7,and also at pH's in excess of about 8.0. It is for this reason that theglucose (and the pH levels determined thereby) levels are desirablymaintained within this range.

The progress of fermentation may be monitored by measuring variousparameters indicative of bacterial growth and toxin production either insamples aseptically removed from the culture vessels or by directmeasurement in the fermentation broth. For example pH may be monitoredwithin the fermentation broth by means of a pH probe. Glucose may bemonitored by a variety of methods known in the art, either directly orindirectly, for example methods described in clinical Diagnosis andManagement by Laboratory Methods, 18 Edn, John Bernard Henry, Editor, WBSaunders Company, Philadelphia, 1991. Examples of direct measurementinclude sugar assays for example those based on chemical reactions suchas for example enzymic reactions, for example reactions based on the useof glucose oxidase, such as calorimetric reactions. Examples includemeasurement by means of dipsticks for example glucose chemstrip BG fromBoehringer Mannheim and use of an on-line glucose monitor. Since pH hasbeen shown to decrease in proportion to glucose consumption, glucose mayalso be monitored indirectly by monitoring pH of the broth. Toxinproduction may be monitored in a variety of known ways, such as forexample, SDS PAGE (Laemmli, Nature 227, 680-684, 1970), ELISA (Nielsenet al, Journal of Clinical Microbiology, 25, 1280-1284, 1987) or anADP-ribosylation assay (Blanke et al., Biochemistry 33, 5155 (1994) orby a combination of these methods. Generally, glucose addition willcommence when the level of glucose in the medium drops to levels suchthat, but for the addition of glucose to generate acidic byproducts,acid would need to be added to control the pH of the fermentationmedium. Generally, additional glucose may be added when the level ofglucose in the fermentation medium drops below about 10 g/L, preferablybelow 2 g/L and more particularly below about 1 g/L. At this level, theexponential growth phase of the culture is extended and the pH of themedia decreased by increased metabolism of glucose. In this way, glucosefeeding may be used to control pH and stabilise the toxin as well as tomaintain growth of culture to obtain high cell density.

We have thus found that with the glucose fed batch fermentation methodaccording to the invention, in a culture medium comprising 10-15 g/Lglucose, the exponential phase of bacterial growth commences generally 5hours after the start of fermentation, with toxin production startingwithin approximately 9 hours after the start of fermentation. Withperiodic addition of glucose commencing generally 8 hours after thestart of fermentation, such as about 9 hours, for example when theculture is in the last 25% increase in growth curve OD value, we havebeen able to achieve toxin production for at least 13.5 hours. In such afed batch method, additional glucose in the range of 7 g/L to 15 g/L maybe consumed.

By the fed batch glucose feeding method of the invention, we have beenable to achieve a cell density in the range of 45-60 (OD at 590 nm) anda toxin production in the range of 145-165 mg/ml culture.

In addition to a carbon source, there are other minimum nutritionalrequirements for growth of C. diphtheriae. These include trace metals,phosphate and a nitrogen source. Generally casamino acids and yeastextract are included in bacterial growth media to provide a nitrogen andamino acid source. Growth media for C. diphtheriae, such as CY medium(Fass et al., Applied Microbiol. Biotechnol. 43, 83-88 (1995)) generallycontains at least 2% yeast extract. We have however found thatincreasing the carbon to nitrogen ratio by reducing the amount of yeastextract below this conventional level, to between 0.5-1.5% such asbetween 0.75 to 1% for example around 1% yeast extract, improves the fedbatch fermentation method according to the invention. Thus we have foundthat reducing the conventional amount of yeast extract results in asignificant improvement in yield of toxin. We have also found animprovement in yield can be obtained by using cystine as opposed tocysteine which can be used in bacterial growth media. Thus a 5-foldincrease in yield of CRM107 has been observed when using 1% yeastextract and using a medium containing 0.7 g/l cystine as compared to 2%yeast extract in a medium containing 0.26 g/l cysteine in a 10 litrefermentation according to the invention.

Accordingly, the use of a growth medium containing no more than 1% yeastextract constitutes a preferred aspect of the method of the invention.

We have found that the reduced yeast extract fermentation conditionsalso have other benefits in addition to increased toxin yield indiphtheria toxin production. Thus as will be described in more detailbelow, reducing the yeast extract content of the growth medium hasadditional benefits in purification of the toxin.

The present invention also relates to a method for purifying diphtheriatoxin from a culture supernatant of a toxin producing bacterial strain.

Diphtheria toxin is secreted in large quantities from synthesisingbacteria such as corynebacteria; it can reach up to 70% of the totalprotein in the culture medium. This level is sufficiently high that forsmall, laboratory scale cultures, a straightforward precipitation forexample using ammonium sulphate or trichloroacetic acid of the culturemedium may be effective alone to purify the toxin. However, difficultiesare encountered when using large scale cultures. The precipitation stepsare time consuming and due to difficulties in handling and dialysingsuch large volumes of material, there is loss of toxin product. We havenow developed a purification method which is capable of handling largevolumes of material and overcomes these disadvantages.

Thus viewed from a further aspect, the present invention provides amethod of purifying diphtheria toxin from a culture of toxin producingbacteria, said method comprising contacting a toxin containingpreparation with an ion exchange matrix, eluting a fraction containingthe toxin, applying the eluate to a hydrophobic matrix, and eluting afraction containing the toxin.

The starting material for purification may for example be a toxincontaining supernatant, a toxin containing cellular fraction or a toxincontaining preparation derived from a culture supernatant, for example aconcentrated supernatant, such as an ultrafiltered supernatant, or adiafiltered supernatant.

In toxin purification methods known in the art, the ion exchange step isperformed after the hydrophobic interaction chromatography step, becausethe art taught that culture supernatant cannot be directly applied to anion exchange matrix, such as DEAE cellulose. Such matrices are capableof binding proteins only under conditions of low ionic strength, and ithas thus been thought that the ionic strength of culture medium would betoo high to achieve efficient binding (Rappuoli et al, J.Chromatography, 268, 543-548, 1983).

We have however found that contrary to this perception in the art, it isindeed possible to apply a culture supernatant onto an ion exchangematrix, such as immobilised DEAE. Preferably, because toxin generallybinds to ion exchange media at low ionic strength, the culturesupernatant is diafiltered prior to applying to the matrix, for examplein the form of a column. This has enabled the two chromatographicseparation steps to be operated in the sequence of ion exchangechromatography (IEC) before hydrophobic interaction chromatography(HIC). In addition, diafiltration may serve as a purification step.

Thus viewed from another aspect, the present invention provides a methodof purifying diphtheria toxin from a culture of toxin producing bacteriasaid method comprising chromatographic steps of ion exchangechromatography and hydrophobic interaction chromatography, characterisedin that said method comprises carrying out an ion exchangechromatography before hydrophobic interaction chromatography.

We have found that this purification method according to the inventionresults in a higher toxin yield as compared to the conventional process,without sacrifice to purity (i.e. without significant levels ofcontaminating proteins). Thus we have found that by carrying out an IECstep before HIC, a purity of up to around 98% can be achieved.

Preferably, the starting material is a culture supernatant for example aculture supernatant from a culture which is fermented using glucose as acarbon source in accordance with the invention. As mentioned above, theuse of lower amounts of yeast extract than is conventional at least inglucose culture media, for example about 1% has additional advantagesbesides improved yield. Thus we have observed that yeast extractcontributes to pigmentation of the growth medium, contributing tocontaminants which can make the subsequent purification process lesseffective. Accordingly, the ability to reduce the levels of contaminantsat the outset represents an advantage for subsequent purification.Culture media with lower yeast content are less pigmented, andaccordingly advantageous.

Thus viewed from a further aspect, the present invention provides amethod of purifying diphtheria toxin comprising

-   (1) fermenting a microorganism strain capable of producing    diphtheria toxin using glucose as a carbon source, said method    comprising adding glucose to a growing culture whereby the addition    of glucose maintains microorganism growth effective to support    diphtheria toxin production; and-   (2) purifying the diphtheria toxin from the culture by contacting a    toxin containing preparation derived therefrom with an ion exchange    matrix, eluting a fraction containing the toxin, applying the eluate    to a hydrophobic matrix, and eluting a fraction containing the    toxin.

Preferably, the microorganism is a bacteria, such as C. diphtheriae. Inthe case of C. diphtheriae, where toxin is secreted into the culturesupernatant, the method may be carried out directly on culturesupernatant or on a preparation derived therefrom such as for example adiafiltered culture supernatant. Thus a preliminary step may involve aprimary clarification of the culture broth to obtain a toxin-containingculture supernatant. Thus bacteria may be separated from the culturebroth by methods known in the art, such as centrifugation or filtration,for example, ultrafiltration, and the resulting supernatant diafilteredor applied directly to the first matrix, the ion exchange matrix. In thecase of expression in other microorganisms, such as E. coli geneticallymodified with toxin, toxin may be found intracellularly, for example inthe periplasm or cytoplasm. In such cases, primary recovery steps maydepend upon the cellular location. The toxin may be extracted from thecells by methods known in the art, for example as described by Skopes inProtein Purification, Principles and Practice, 3rd edn, Pub: SpringerVerlag, followed by purification in accordance with the method of theinvention.

Filtration to clarify the fermentation broth may be effected by methodsknown in the art, for example with membranes such as hollow fibre orspiral wound membranes, such as by means of a 0.1 or 0.2 μm filter, forexample a hollow fiber filter, such as that obtainable from A/GTechnology, or a 0.4 or 0.65 μm hollow fibre or spiral wound membrane,or a 300K or 500K filter.

For ease of handling, particularly where large volumes are concerned,such as would be the case for ‘industrial’ scale purification forpharmaceutical purposes, a degree of concentration of the supernatantmay be effected prior to the ion exchange step. The cell free culturesupernatant may be concentrated, generally 5 to 50 fold, preferably 15to 25 fold, such as 20 fold, using protein concentration methods knownin the art, for example by means of ultrafiltration with porousmaterials for example in the form of filters, membranes or hollowfibres. For ease of handling, filters are preferred. Forultrafiltration/concentration, filters having a molecular weight cut offsmaller, preferably 20% smaller than the toxin, are preferred,preferably 30K filters (i.e. filters which have a 30000 dalton molecularweight cut off). Suitable materials for such filters are known in theart and include polymeric materials such as mixed cellulose, polyethersulfone or PVDF, for example polysaccharides such as cellulose, andpolysulfones. Preferred materials are those which have a lower capacityor ability to absorb toxin. Cellulose filters are particularlypreferred, for example filters made from regenerated cellulose such asthe ‘YM’ based filters and other membranes which have little proteinbinding capacity for example the Flat plate tangential flowbioconcentrators produced by Amicon. The use of cellulose filters forultrafiltration thus constitutes a preferred aspect of the purificationmethod according to the invention.

IEC may be carried out directly on the culture supernatant, using anappropriately sized bed volume as determined by one skilled in the art.Optionally, the concentrated clarified supernatant may be furthertreated prior to chromatographic purification, for example bydiafiltration. In this way, ionic strength may be reduced by removingsalts and other ions smaller than the molecular weight cut off size ofthe diafiltration membrane. The reduction in ionic strength has benefitsfor the ensuing ion exchange step, since at reduced ionic strength, lesstoxin is retained by the ion exchange matrix and yield is thus improved.Furthermore, a partial purification is achieved by the diafiltrationmembrane. Diafiltration may thus be carried out against a low ionicstrength buffer, for example Tris, Tricine, MES, Bis-Tris, TES, MOPS andphosphate, in a concentration of from about 0.1 mM to about 100 mM,preferably from about 10 to about 50 mM, for example 10 mM, having a pHof from about 5 to about 8, preferably from about 6 to about 8 forexample about 7.4 to 7.6, using for example a 30 000 cut off membranewhich will result in removal of salts, low molecular weight mediacomponents and secreted proteins less than 30 000 dalton molecularweight. Such components may otherwise bind to the ion exchange matrixand reduce its capacity to bind toxin. Diafiltration may be carried outfor example using materials of the type used for the ultrafiltrationstep, against buffers such as low ionic strength buffers such as Tris,Tricine, MES, BiS-Tris, TES, MOPS or phosphate, in a concentration offrom about 0.1 mM to about 100 mM, preferably from about 10 to about 50mM, for example 10 mM, having a pH of from about 5 to about 8,preferably from about 6 to about 8 for example about 7.4 to 7.6, forexample 10 mM potassium phosphate pH 7.6 so as to reduce theconductivity of the concentrated culture supernatant as low as possible,preferably below about 5 mS/cm.

Treatment of the culture supernatant in this way prior to IEC thusovercomes the problems of the prior art method which requires that HIChas to take place before IEC. The combination of diafiltration andultrafiltration not only reduces the ionic strength, but serves as aninitial purification and allows the volume to be reduced. This meansthat smaller columns may be used, thereby reducing the time required forthe chromatographic steps to be carried out.

A further advantage is that toxin purification according to the methodof the invention is faster overall than the conventional method, thuslimiting the time during which the toxin is exposed to room temperatureand vulnerable to degradation.

The chromatographic steps may be carried out using ion exchange orhydrophobic matrices as appropriate in batch or column form, the latterbeing preferred for both speed and convenience. The matrix may be aconventional support as known in the art for example inert supportsbased on cellulose, polystyrene, acrylamide, silica, fluorocarbons,cross-linked dextran or cross-linked agarose.

Any conventional ion exchange resin may be used. Examples include Qsepharose and diethylaminoethyl (DEAE) and quaternary amine resins. Theanion exchange material may be packed into a column, whose size will bedependent upon the volume of culture supernatant to be used. Theappropriate column size may be determined by those skilled in the artaccording to the total protein in the concentrated media. Generally, forlarge scale cultures of the order of 40-50 litres, 2 litre supernatantconcentrates may be applied to columns of volume 1.25 l. The column mayfirst be equilibrated with a buffer for example a low ionic strengthbuffer such as Tris, Tricine, MES, Bis-Tris, TES, MOPS or phosphate, ina concentration of from about 0.1 mM to about 100 mM, preferably fromabout 10 to about 50 mM, for example 10 mM, having a pH of from about 5to about 8, preferably from about 6 to about 8 for example about 7.4 to7.6, for example the buffer used to diafilter the concentratedsupernatant, such as 10 mM potassium phosphate pH 7.6. The culturesupernatant or concentrate may then be loaded, the column washed with abuffer of low ionic strength and the same pH as the equilibration bufferto wash off any unbound protein, for example the equilibration buffer.

Bound toxin may then be eluted in a variety of ways. These includealtering the pH or increasing the ionic strength of the buffer. Thustoxin may be eluted by a gradient increase of buffer with high ionicstrength, such as Tris, Tricine, MES, Bis-Tris, TES, MOPS or phosphate,in a concentration of from about 10 mM to about 1.0 M, preferably fromabout 10 mM to about 500 mM, containing salts like NaCl, KCl or ammoniumsulphate at a concentration of from about 0.1M to 1.0M. These buffersmay have a pH of from about 5 to about 8, preferably from about 6 toabout 8 for example about 7.0 to 7.6. One example of a preferred bufferis 10 mM potassium phosphate containing 500 mM KCl at pH 7.6. Theprotein will be eluted between 100 to 150 mM KCl in the buffer.

The toxin containing eluate may then be applied to the hydrophobicmatrix. Optionally but preferably, the ionic strength of the eluate maybe increased prior to the second hydrophobic interaction step, by mixingwith a buffer of appropriate ionic strength or by diafiltration. Thismay facilitate binding of toxin to the hydrophobic resin. Thus theeluate may be mixed with a high ionic strength buffer for example Tris,tricine, MES, Bis-Tris, TES, MOPS or phosphate, in a concentration offrom about 10 mM to about 1.0M, preferably from about 10 mM to about 500mM, containing salts like NaCl, KCl or ammonium sulphate at aconcentration of about 0.1M to 1M. The buffers may have a pH of fromabout 5 to about 8, preferably from about 6 to about 8 for example about7.0 to 7.6. One example of a preferred buffer is 10 mM potassiumphosphate buffer containing 500 mM ammonium sulphate with a pH of 7.0.Diafiltration may be carried out using methods known in the art, forexample using ultrafiltration membranes, such as 30K or 10K cut offhollowfibre filters obtainable from A/G technology or spiral woundfilters from Amicon, or Ultrasette (Omega) (Pall Filtron) and usingbuffers such as the aforementioned tris, phosphate, acetate or HEPES.

Hydrophobic matrices are known in the art. These include theaforementioned supports carrying hydrophobic moieties such as alkyl, forexample butyl, hexyl, octyl, acetyl or phenyl groups, for example alkylagarose such as decyl agarose.

The hydrophobic matrix material may be packed into a column, whose sizewill be dependent upon the volume of culture supernatant to be used. Theappropriate column size may be determined by those skilled in the art.Generally, for large scale cultures of the order of 40-50 litres, theeluate from the IEC step will be generally in a volume of 11 to 2 l andwill be applied to columns whose size may be determined by those skilledin the art according to the amount of protein, but may be of the orderof 250 ml for protein concentration of up to 100 mg/ml. The column mayfirst be equilibrated with a high ionic strength buffer, for exampleTris, Tricine, MES, Bis-Tris, TES, MOPS or phosphate, in a concentrationof from about 10 mM to about 1.0 M, preferably from about 10 mM to about500 mM, containing salts like NaCl, KCl or ammonium sulphate at aconcentration of about 0.1M to 1.0M, having a pH of from about 5 toabout 8, preferably from about 6 to about 8 for example about 7.6, forexample the buffer used to diafilter the concentrated supernatant or thebuffer mixed with the eluate, such as for example 50 mm potassiumphosphate with 1M ammonium sulphate pH 7.0. The IEC eluate, or eluatemixed with high ionic strength buffer or diafiltered eluate may then beloaded, the column washed with a high ionic strength buffer for exampleTris, Tricine, MES, Bis-Tris, TES, MOPS or phosphate, in a concentrationof from about 10 mM to about 1.0 M, preferably from about 10 mM to about500 mM, containing salts like NaCl, KCl or ammonium sulphate at aconcentration of about 0.1 M to 1.0 M, having a pH of from about 5 toabout 8, preferably from about 6 to about 8 for example about 7.6, suchas for example the column equilibration buffer to remove any unboundproteins. Bound toxin may then be eluted in a variety of ways forexample by gradient increase of a polar solvent in the wash buffer or bygradient increase of a low ionic strength buffer, for example Tris,Tricine, MES, Bis-Tris, TES, MOPS or phosphate, in a concentration offrom about 10 mM to about 1.0 M, preferably from about 10 mM to about500 mM, containing salts like NaCl, KCl or ammonium sulphate at aconcentration of about 0.1M to 1.0M, having a pH of from about 5 toabout 8, preferably from about 6 to about 8 for example about 7.6, forexample 50 mM phosphate containing 1M ammonium sulphate pH 7.0 to 50 mMphosphate buffer pH 7.0 without ammonium sulphate. Toxin may be elutedin such a gradient as the ammonium sulphate concentration is reduced toapproximately 700 mM.

By using the method of the invention, we have been able to purifydiphtheria toxin mutant CRM107 from large volumes of culturesupernatant, of the order of 50 l with an average yield of 32% of thestarting material and purity greater than 98% as measured by HPSEC (highperformance size exclusion chromatography). This for the first timeenables the properties of diphtheria toxin mutants such as the bindingmutant CRM107 to be exploited for preparing therapeutic products.

When the purified toxin is a mutant toxin to be used to prepare targetedtoxin derived therapeutic agents, the toxin may conveniently be elutedwith a buffer suitable for carrying out the conjugation or attachment ofthe toxin with a cell specific binding or targeting moiety, for examplea cell recognition moiety such as an antibody to a cell surface moietyor an antigen binding fragment thereof or a protein which has a bindingpartner on the cell surface for example in the form of a receptor, suchreceptors having some degree of selectivity, i.e. being present on somebut not all cell types.

As used herein, ‘cell specific’ or ‘cell selective’ refers to a moietywhich has a targeting or binding affinity for a cell surface moietywhich is not present on all cells, and thus which is selective forcertain cells or groups or types of cells or specific receptors. Inother words, it encompasses a moiety which enables a diphtheria toxin ormutant toxin conjugated to it to be targeted selectively.

Thus according to a further aspect, the present invention provides aprocess for preparing a diphtheria toxin conjugate comprising linking adiphtheria toxin produced by the method of the invention with a cellspecific binding or targeting moiety.

Examples of cell specific moieties include antibodies to moietiesexposed on the surface of particular cells, and proteins such as growthfactors or transferrin whose binding partners in the form of receptorsare expressed only on particular cell types, or predominantly only onspecific cell types.

Conjugation may be effected by methods known in the art such as chemicalcross-linking or covalent bond formation. Preferably the conjugation isbe means of covalent bond formation for example between maleimido groupsintroduced onto one component of the conjugate and thiol groupsintroduced onto the other. In such a method, one of the two componentsis modified by introduction of maleimide groups and the other ismodified by means of introduction of thiol groups. Preferably thediphtheria toxin or mutant is modified by means of maleimide groups andthe cell specific targeting moiety by means of thiol groups. Preferablyfor the preparation of anticancer agents such as those for the treatmentof malignant glioma, the cell selective moiety is transferrin sincetransferrin receptors are expressed in quantity in rapidly dividingcells such as glioma cells but are essentially absent on other cells inthe CNS. In such conjugates, generally the diphtheria toxin element ismodified with maleimide and the transferrin element with thiol groups.However, diphtheria toxin element may be modified with thiol groups andthe transferrin element with maleimide.

Conventional modifying agents known in the art may be used. Examplesinclude esterifying compounds, thiol activating compounds and carboxylmodifying agents such as N ethyl maleimide andmaleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) and 2-iminothiolane.In a typical conjugation, MBS may be added to diphtheria toxin in aratio of from 1 to 100 times molar excess, such as 2 to 5 preferably 3.5times, incubated at a temperature of 2 to 50° C. for example 15 to 20°C. such as room temperature, for 5 minutes to 24 hours, such as from 20to 40 minutes such as 30 minutes followed by removal of excess reagentby techniques known in the art such as gel filtration for example usingSephadex G-25 desalting. The eluate may then be cooled prior to theconjugation reaction. Crude intermediates may be removed prior toconjugation by methods known in the art such as precipitation, dialysis,chromatography, extraction. Transferrin may be thiolated by means ofincubation with 2-iminothiolane in a ratio of 1 to 100 times molarexcess, such as 5 to 10 times molar excess preferably 7 to 8 times suchas 7.7 times at a temperature of 2 to 50° C., for example 35-40° C., for5 minutes to 24 hours, such as 20 to 40 minutes such as 30 minutesfollowed by removal of excess reagent by techniques known in the artsuch as gel filtration. For the conjugation, the two functionalisedreagents may be mixed in a ratio of 1 to 2 preferably at a temperatureof 2 to 8° C. for 4 to 24 hours, preferably 12 to 20 hours, for example18 hours.

According to a yet further aspect, the present invention provides amethod of treatment of a CNS neoplasm comprising administering to asubject a diphtheria toxin conjugate produced by the method of theinvention.

According to a still yet further aspect, the present invention providesthe use of a diphtheria toxin conjugate produced by the method of theinvention in the manufacture of a medicament for use in the treatment ofCNS neoplasm.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A shows the growth results in batch fermentation lots, using 0.8%glucose/2.4% maltose (A), 2.4% glucose (B) and 1.5% glucose/2.4% maltose(C).

FIG. 1B shows the growth results of batch fermentation lots in mediacontaining 2% yeast abstract and 0.26 g/l cysteine (D) and 1% yeastextract and 0.7 g/l cystine (E).

FIG. 2 shows CRM107 concentration post fermentation with conditions of(A), (B) and (C) measured by SDS-PAGE.

FIG. 3 shows CRM107 concentration post fermentation with conditions of(D) and (E) measured by ELISA.

The invention will now be described with reference to the followingnon-limiting examples.

EXAMPLE 1 Comparison of Batch and Fed Batch Growth

This study was carried out in a 10-L fermenter. Three batchfermentations were investigated, using 0.8% glucose/2.4% maltose (A),2.4% glucose (B) and 1.5% glucose/2.4% maltose (C). Two fed-batchprocesses were investigated, using 1.5% glucose as the startingconcentration with a glucose feed, in media containing 2% yeast extractand 0.26 g/l cysteine (D) and 1% yeast extract and 0.7 g/l cystine (E).

Methods Media Preparation

The composition of the five media analysed are shown in Table 1, showingvariations in the type and amount of the carbon source, the amount ofyeast extract and substitution of cysteine for cystine. The media werebased on NIH medium (which comprises 20 g/L yeast extract, 20 g/Lcasamino acids, 5 g/l KH₂PO₄, 15 g/L glucose, 0.7 g/l cystine, 0.1 g/Ltryptophan, 1.5 mg/L beta alanine, 1.5 mg/L nicotinic acid, 0.075 mg/Lpimelic acid, 0.03 ml of 10 mM HCl, and 10 ml/l metals supplementsolution at pH 7.4).

Strain

All examples were carried out using Corynebacterium diphtheriae (β tox⁺)producing CRM107 Phe³⁹⁰ Phe⁵²⁵ as described by Greenfield et al, Science238, 536-539, 1987.

Inoculum Preparation

The inoculum for (A) was grown in 100 mL of LICY medium (Mueller et al,J. Immunol 40, 21-32, 1941) in 500 mL flasks, supplemented with glucoseat the concentration of 0.8% (w/v). The cultures were incubated at 34±2°C. at 300±50 rpm for 14.5 hours, at which time the OD₅₉₀ was 11.5 andthe fermenter was inoculated to a 1.5% (v/v) inoculum.

The inoculum for (B) and (C) was prepared as follows: Two vialscontaining 1.0 mL of a glycerol frozen culture of Corynebacteriumdiphtheriae (β tox⁺), were inoculated into a 500 mL flask containing 100mL±10.0 mL of sterile LICY (NM5) media and incubated at 34±2° C. at300±50 rpm. The above was carried out in triplicate, which included aback-up inoculum culture. After 15 hours of incubation, the opticaldensity and a Gram stain of the shake flask contents were performed. Theoptical density at 590 nm of the pooled cultures was 3.4 and 3.9. TheGram stain showed Gram positive rods that were mostly club shaped,indicative of C. diphtheriae. The fermenters were inoculated to a 5%(v/v) inoculum with these pooled cultures.

The inoculum for (D) and (E) were prepared as follows: Two vialscontaining 1.0 mL of a glycerol frozen culture of Corynebacteriumdiphtheriae (β tox⁺), were inoculated into each of 2.8 L Fernbach flaskcontaining 250 mL±10.0 mL of sterile LICY (NM5) media without a carbonsource in duplicates, and incubated at 34±2° C. at 300±50 rpm. One ofthe flask contained cysteine with 2% yeast extract (for the inoculationof D) and the other contained cystine with 1% yeast extract (for theinoculation of E). Additional inocula for the two fermentations wereprepared in three 500 mL flasks each, to enable a pooled inoculum sizeof 5% (v/v) into the production fermenter. These were prepared from 300μL of glycerol frozen culture of Corynebacterium diphtheriae (β tox⁺),were inoculated into a 500 mL flask containing 100 mL±2.0 mL of sterilemedia identical to the fermentation media without the carbon source, andwere incubated at 34±2° C. at 300±50 rpm. Back-up inoculum cultures wereprepared in triplicate in 500 mL flasks as above but in media containing1.5% glucose. After 15 hours of incubation, the optical density and aGram stain of the shake flask contents were performed. The opticaldensity at 590 nm of the cultures was 3.01 and 1.62 for D and E,respectively. Gram stain showed Gram positive rods that were mostly clubshaped, indicative of C. diphtheriae. The fermenters were inoculated toa 5% (v/v) inoculum with these.

In-Process Monitoring

Samples were taken at one hour intervals. Every hour, the date, actualtime, temperature, pH, and agitation were recorded. Residualfermentation medium glucose concentration and the optical density at 590nm of the fermentation broth were also determined on an hourly basis.

Optical Density and Residual Glucose Concentration

Optical density was measured using the Pharmacia Ultrospec 4000UV/Visible spectrophotometer set at 590 nm. Milli Q water was used toset the reference blank and for diluting the culture as required.Residual glucose concentration was estimated with a glucose Chemstrip BGfrom Boehringer Mannheim.

Temperature, pH, RPM and Aeration

Temperature and pH were measured directly inside the fermenter using aPT100 temperature probe and a Bradley James pH probe, respectively. RPMwas measured directly off the agitation shaft of the fermenter. Aerationwas measured using a rotameter.

Fermenter Harvest (Recovery of Supernatant)

Approximately 2 L of clarified fermentation broth (A) was obtained bycentrifugation. This method was used because of the excessive amounts ofantifoam required during the fermentation. For (B), (C), (D) and (E) 5 Lof the fermentation broth was clarified by microfiltration, using the0.2 micron A/G Technology hollow fiber filter. Before filtration wasstarted, the medium was cooled down to about 10° C. Once cross-flow wasestablished, the filtrate clamp was slowly released to give a filtrateflow of about 700 mL/min. The retentate was pumped back into thefermenter, while 5 L of the filtrate were collected in sterile bottlesand stored immediately at −70° C.

SDS-PAGE, ELISA and ADP Ribosylation Activity Analyses

Time-course samples of the fermentation were immediately frozen in 2 mLaliquots in cryo vials at −70° C. and subsequently analysed by SDS-PAGEto confirm the presence of CRM107. Samples showing the CRM107 proteinband were scanned with a Sharp JX-330 scanner to estimate theconcentration of CRM107 in the fermentation broth. CRM107 concentrationwas also analysed by sandwich ELISA and ADP ribosylation activity wasmeasured.

Results and Discussion

In this set of experiments, five different media experiments werecompared, including the 0.8% glucose/2.5% maltose (A), 2.4 glucose (B),1.5% glucose/2.4% maltose (C), 1.5% glucose (with cysteine; D), 1.5%glucose (with 1% yeast extract, E).

Growth of Corynebacterium diphtheriae

As shown in FIG. 1 a, C. diphtheriae grew to a final OD₅₉₀ of 25 to 28in 14 hours in the three batch fermentation processes. Growth wasinitially slower in the 2.4% glucose medium, which might have been dueto a ‘culture shock’ following transfer of the innoculum from a mediumwithout carbon source. Growth of C. diphtheriae in the two fed-batchfermentation processes was faster in the glucose+cysteine medium than inthe glucose+cystine medium. However, the growth rate as well as thefinal OD₅₉₀ were essentially the same (FIG. 1 b).

Production of CRM107

The production of CRM107 during the course of the fermentation wasanalysed both by SDS-PAGE, ELISA and ADP-ribosylation assay. In thethree batch processes, the 1.5% glucose/2.4% maltose medium supported ayield of 5 S mg/L (ELISA data) after 12 hours of fermentation (FIG. 2),which was the highest production achieved for the Batch processes. The2.4% glucose medium was trailing behind in both growth and CRM107production, by approximately 4 hours, consequently the production wasdelayed. The long lag phase may be due to the culture shock due to thehigh glucose concentration in the medium, particularly when the inoculumwas grown in the absence of a carbon source. Despite this delayproduction began at 12 hours and continued to 13 hours, and could haveperhaps continued a while longer. This is evidenced by the continuedincrease in CRM107, concentration observed in the 0.2 μm fraction (seeTable 1), reaching 47 mg/L (ELISA data), as the fermenter was allowed torun for an additional hour while the other fermenter was beingharvested. The 0.8% glucose/2.4% maltose medium produced very littleCRM107, despite that the cell yield was equivalent to that obtained withthe other fermentation media. This may be due to excessive foamingencountered with this fermentation, which resulted in not only a highconsumption of antifoam but also forced the dissolved oxygen to drop tonearly 0 for a 15 to 20-minute period, as the agitation was turned downto less than 100 rpm, may have played a role in the observed poor yield.

The CRM107 yield is significantly higher than that obtained fromglycerol fermentation (data not shown), with an increase ofapproximately 14-fold in the post-fermentation CRM107 concentration asmeasured by SDS PAGE.

In the fed-batch processes, the 1.5% glucose medium, with half theamount of yeast extract used in the other four media, proved to be thebest overall medium for CRM107 production. The final yield approached100 mg/L of CRM107 in 12 hours of fermentation (FIG. 3). Glucose feedingstarted at about 9.5 hours of fermentation and proceeded for 3.5 hours.A total of 125 g of glucose (251 mL of a 50% solution) was added to thefermenter as a means of controlling the pH, when acid was required laterduring the course of the fermentation (Table 2). The strategy forfeeding glucose relied on the adjustment of pH by organic acids producedfrom glucose metabolism, instead of using inorganic acid.

We also analysed CRM107 concentration by the ADP-ribosylation assay. Theresults are shown in Table 3, comparing the concentration of CRM107determined by ELISA and ADP-ribosylation assay. The product obtained isshown in the ADP-ribosylation assay to be biologically active. Glucosefed-batch (1.5% glucose) containing 1% yeast extract results in optimumtoxin production.

Scale Up

We have scaled up this process based on the optimum conditions of fedglucose, 1% yeast extract to 50 l and observed that exponential growth,as in the 10 l culture, started around 6 hours and continued until 13hours. After 14 hours, C. diphtheriae grew to an OD₅₉₀ of 37.4. CRM107production as judged by SDS PAGE was 165.5 mg/l at the end offermentation.

TABLE 1 Summary of the five optimisation fermentation processes Lot A BC D E Fermentation Process Batch Batch Batch Fed-Batch Fed-Batch Carbonsource 0.8% Glucose/2.5% 2.4% Glucose 1.5% Glucose/2.4% 1.5% Glucose1.5% Glucose Maltose Maltose Yeast extract 20 g/L 20 g/L 20 g/L 20 g/L10 g/L Cystine/Cysteine Cystine Cystine Cystine Cysteine Cystine 0.7 g/L0.7 g/L 0.7 g/L 0.26 g/L 0.7 g/L Initial pH 7.21 7.17 7.22 7.17 7.24Final pH 7.21 7.16 7.21 7.19 7.22 Final OD 25.4 27.8 26.8 45.3 45.3CRM107 concentration post fermentation 0 mg/L 18.0 mg/L 44.0 mg/L 11.0mg/L 98.0 mg/L measured by SDS-PAGE CRM107 concentration 0.83 mg/L 28.7mg/L 36.9 mg/L 9.13 mg/L 63.6 mg/L post fermentation, measured by ELISACRM107 concentration 0.86 mg/L 32.72 mg/L 70.88 mg/L post fermentation,measured by ADP-ribosylation assay CRM107 concentration ND 25.0 mg/L48.0 mg/L 11.0 mg/L 90.0 mg/L Post microfiltration, measured by SDS-PAGECRM107 concentration ND 47.0 mg/L 29.9 mg/L 4.73 mg/L 108.8 mg/L Postmicrofiltration, measured by ELISA CRM107 concentration ND 84.76 mg/L90.76 mg/L 154.9 mg/L Post microfiltration, measured by ADP-ribosylationassay

TABLE 2 Glucose feed during the Fed-batch fermentation of C.diphtheriae, Lots #D and E Cumulative Amount of Glucose Fermentationadded (g/fermenter) Time (h) D E 8.00 0.0 0.0 8.17 0.0 0.0 9.00 0.0 9.09.50 40.5 54.0 10.00 95.0 107.5 11.00 105.5 125.5

TABLE 3 ADP-ribosylation activity of CRM107 in the fermentation brothtime course samples Lot #A, B and C Fermen- CRM107 CRM107 CRM107 Fermen-tation Concentration Concentration Concentration by tation Lot by ELISAby SDS-PAGE ADP-Ribosylation Samples Number (mg/L) (mg/L) (mg/L) T = 12A 1.2 Und⁽¹⁾ 3.21 T = 14 A 0.8 Und⁽³⁾ 0.86 T = 12 B 17.4 ND 12.36 T = 14B 28.7 18.0 32.72 0.2 μm B 35 25.0 84.76 T = 12 C 55.5 47.0 69.72 T = 14C 36.9 44.0 70.88 0.2 μm C 29.3 48.0 90.7 Und⁽¹⁾: Undetectable.

EXAMPLE 2 Primary Recovery of CRM107 Produced from a Fermentation ofCorynebacterium diphtheriae (β Tox^(±))

CRM107 was recovered from fermentation broth by two steps. The firstaims at clarifying the fermentation broth, using a 0.1 μm or 0.2 μm A/GTechnology hollow fiber filter. The second step achieves an approximate10-fold concentration of the cell-free fermentation broth, using a 30KA/G Technology hollow fiber membrane. This concentrated clarifiedfermentation broth is then used as the starting material for thepurification of CRM107 (see Example 3).

This study investigates a number of recovery processes using the 0.1 μmfiltrate from a 50-L glucose fed-batch fermentation as the startingmaterial. The objective was to develop a process that minimizes productloss during primary recovery.

Procedure Materials

The various filters tested in this study are listed in Table 4. Theywere tested for their efficiency during the ultrafiltration anddiafiltration processes.

TABLE 4 Types of filters tested in the primary recovery optimizationstudy Membrane types Cat # Characteristic AMICON MiniPlate-10 157130310,000NMWC flat plate AMICON MiniPlate-30 1571304 30,000NMWC flat plateFILTRON 10K Alpha AS010C72 10,000NMWC flat plate AIG Technology 1 OKUFP-10-C-6A 10,000NMWC hollow fiber AIG Technology 30K UFP-30-C-6A30,000NMWC hollow fiber FILTRON 10K Omega 0501070 10,000NMWC flat plateseries

Fermentation Broth Clarification

The fermentation broth used to optimize the primary recovery of CRM107was obtained from a 50-L glucose fed-batch fermentation of C.diphtheriae (β tox⁺), as in Example 1. Twenty liters of the fermentationbroth was clarified using the 0.1 μm A/G Technology hollow fiber filter.

Primary Recovery Schemes

Six primary recovery schemes (as summarized in Table 5) were evaluatedusing the 0.1 μm filtrate as the starting material.

TABLE 5 CRM107 primary recovery optimization schemes Primary recoveryoptimization schemes Filtration Scheme Scheme Scheme Scheme SchemeScheme Step # type #1 #2 #3 #4 #5 #6 1 Microfiltration 0.1 μm hollowfibre 2 Ultrafiltration 30K A/GT 30K A/GT 30K 10K 10K 30K A/GT hollowhollow Amicon Amicon Filtron hollow fibre fibre flat flat alpha fibreplate plate flat plate 3 Diafiltration 30K A/GT 10K A/GT 30K 10K 10K 30KA/GT hollow hollow Amicon Amicon Filtron hollow fibre fibre flat flatalpha fibre plate plate flat plate

Primary Recovery Schemes #1 and 2

In scheme #1 12 L of the 0.1 μm filtrate was concentrated to 1.2 L usinga 30K A/GT hollow fiber. This retentate was then divided into two equalbatches of 550-mL, which had a starting conductivity of 16.8 mS/cm. Inscheme #1, one 550-mL batch was diafiltered using a 30K A/GT hollowfiber filter, and the volume kept at approximately 500-mL with constantaddition of 10 mM phosphate buffer. The diafiltration was stopped whenthe conductivity of the retentate was less than 5 mS/cm (4.34 mS/cm). Atotal of approximately 2 L of permeate was collected.

In scheme #2, the other 550-mL batch was diafiltered using a 10K A/GThollow fiber filter, and the volume kept at approximately 500 mL withconstant addition of 10 mM phosphate buffer. Diafiltration was stoppedwhen the conductivity of the retentate was less than 5 mS/cm (3.97mS/cm). A total of approximately 2 L of permeate was collected. The aimof scheme #2 was to minimize CRM107 loss during diafiltration.Diafiltration was to reduce salt interference during later stages ofpurification (such as ion exchange chromatography).

Primary Recovery Schemes #3, 4, 5 and 6

These four schemes were based on the use of Amicon and Filtron flatplate tangential flow filters for the concentration and diafiltrationsteps. Schemes 3, 4 and 6 were to evaluate different membranechemistries.

In scheme #3, 950 mL of the 0.1 μm filtrate was concentrated toapproximately 125 mL using a 30K Amicon tangential flow filter. Theconductivity of the retentate was 18.6 mS/cm. The retentate was broughtup to approximately 200 mL with 10 mM phosphate buffer, and thendiafiltered using the same filter. The volume was kept at approximately200 mL with constant addition of 10 mM phosphate buffer. Diafiltrationwas stopped when the conductivity of the retentate was reduced to lessthan 5 mS/cm (4.34 mS/cm). A total of approximately 600 mL of permeatewas collected. The final retentate volume was 75 mL.

In scheme #4, 550-mL of the 0.1 μm filtrate was concentrated down toapproximately 100 mL using a 10K Amicon tangential flow filter. Theconductivity of the retentate was 18.5 mS/cm. The retentate was broughtup to approximately 200 mL with 10 mM phosphate buffer, and thendiafiltered using the same filter. The volume was kept at approximately200 mL with constant addition of 10 mM phosphate buffer. Diafiltrationwas stopped when the conductivity of the retentate was less than 5 mS/cm(4.43 mS/cm). A total of approximately 500 mL of permeate collected. Thefinal retentate volume was 83 mL.

In scheme #5, 950 mL of the 0.1 μm filtrate was concentrated down toapproximately 150 mL using a 10K Filtron alpha tangential flow filter.The conductivity of the concentrate was 17.2 mS/cm. The retentate wasbrought up to approximately 200 mL with 10 mM phosphate buffer, and thendiafiltered using the same filter. The volume was kept at approximately200 mL with constant addition of 10 mM phosphate buffer. Diafiltrationwas stopped when the conductivity of the retentate was less than 5 mS/cm(3.80 mS/cm). A total of about 800 mL of permeate was collected. Thefinal retentate volume was 72 mL.

In scheme #6, approximately 18 L of the 0.1 μm filtrate was concentratedto approximately 1.8 L using a 30K hollow fiber filter. The retentatewas diafiltered using a 10K omega Filtron tangential flow membrane. Thevolume was kept constant at approximately 400 mL with constant additionof 10 mM potassium phosphate buffer. The diafiltration was stopped whenthe conductivity was less than 5 mS/cm (4.74 mS/cm).

Primary Recovery Sample Analysis

The primary recovery optimization was using 1.8 mL samples taken at thestart and end of the process for analysis of CRM 107 recovery andprocess efficiency. Samples were immediately frozen in cryogenic vialsat −70±5° C. and subsequently analyzed by SDS-PAGE, isoelectricfocussing, Western blot, and HP-SEC.

SDS-PAGE and Western Blot

SDS-PAGE and Western blot analysis of the samples were carried on aBio-Rad precast 4-15% polyacrylamide gradient gel. Samples were diluted1-2 fold in phosphate buffered saline (for the permeate and 0.1 μmfiltrate samples), or 20 fold (for the retentate), and then anadditional two fold in the solubilization buffer solution. Twentymicroliters of the mixture were loaded on a gel, and electrophoresed at200 V for 50 min. Samples showing the CRM107 protein band on SDS-PAGEwere scanned with a Sharp JX-330 scanner to estimate the concentrationof CRM107. For Western blot analysis, the protein bands on thepolyacrylamide gel were transferred onto a nitrocellulose membrane andprobed with mouse anti-human diphtheria toxin, and detected withanti-mouse IgG alkaline phosphatase conjugate.

Isoelectric Focussing (IEF)

IEF analysis of the retentate samples was carried out using thePharmacia Ampholine PAG plate (pI 4-6.5), with pI markers ranging from3.6-6.6. Samples were appropriately diluted between 1 to 5 fold. Twentymicroliters of the mixture were loaded on a gel, and electrophoresed ata voltage of 2000 V, at 25 mA, for 2.5 h.

High Performance Size Exclusion Chromatography (HP-SEC)

The final samples of the recovery optimization process, obtained afterthe diafiltration step, were analyzed by HP-SEC to evaluate the degreeof purity of the product and the impact of the process on pigmentremoval. One hundred microliters of the samples were loaded on thecolumn and the chromatograph monitored at a wavelength of 280 nm. TheCRM107 peak eluted with a retention time of about 9.3 min. The % peakarea was used to estimate product purity.

Results and Discussion

Six optimization schemes of CRM107 primary recovery were evaluated forproduct recovery and process efficiency in facilitating the subsequentpurification of CRM107. The criteria applied for the primary recoverywere (i) to achieve a 5-10 fold concentration of the 0.1 μm-clarifiedfermentation broth with an ultrafiltration step, and (ii) to reduce theconductivity of the concentrated broth to below 5 mS/cm with adiafiltration step.

The optimization study was monitored using four different analyses(SDS-PAGE, Western blot, IEF and HP-SEC). Western blot and IEF datashowed the presence of CRM107 in all retentate samples. Similarly, theSDS-PAGE data showed CRM107 in the retentate samples, as well as in the0.1 μm filtrate sample (Table 6). Analysis of the permeate samples bySDS-PAGE suggest that all schemes tested, except for scheme #1 and 5,did not cause any noticeable loss of CRM107 in the permeate. On thebasis of SDS-PAGE, IEF and HP-SEC data, scheme #3 appears to be theprimary recovery process of choice. The SDS-PAGE data do not supportthis conclusion as the recovery post-diafiltration is good in mostcases, except for scheme #5, where noticeable loss of product wasevident. Data from IEF show more bands in the sample processed by scheme#5, implying that this diafiltered sample was the least clean. In allsamples analyzed by IEF, three prominent bands were visible, identicalto those present in the reference standard sample, with a pI between 5.0and 5.4.

The purity achieved with scheme #3 after this step was 17.5% compared to9% CRM107 purity with scheme #1, Table 4. The recovery of CRM107 is bestwith scheme #3, which showed a CRM107 peak area of 161, nearly 2-foldgreater than that from scheme #1.

TABLE 6 Material balance of the different primary recovery schemes(SDS-PAGE data) Primary Recovery Scheme Filtration Step 1 2 3 4 5 6Microfiltration Filtrate CRM107 conc. (g/L) 0.113 0.113 0.102 0.1130.114 0.113 Filtrate volume (L) 6.0 6.0 0.950 0.550 0.950 18.0 FiltrateCRM107 total amount (mg) 678.0 678.0 96.9 62.3 108.3 2034Ultrafiltration Retentate CRM107 conc. (g/L) 1.055 1.055 1.067 0.3750.445 Retentate volume (L) 0.550 0.550 0.125 0.100 0.150 RetentateCRM107 total amount (mg) 580.3 580.3 133.4 37.5 66.8 Ultrafiltration N/AFiltrate CRM107 conc. (g/L) 0.012 0.012 0 0 0.041 Filtrate volume (L)5.45 5.45 0.825 0.450 0.800 Filtrate CRM107 total amount (mg) 65.4 65.40 0 32.8 Diafiltration Retentate CRM107 conc. (g/L) 1.396 1.197 1.2710.834 0.358 3.429 Retentate volume (L) 0.5 0.5 0.075 0.083 0.072 0.400Retentate CRM107 total amount (mg) 698 598 95.3 69.2 25.8 1372Diafiltration Filtrate CRM107 conc. (g/L) 0 0 0 0 0.031 Filtrate volume(L) 2 2 0.6 0.5 0.8 N/A Filtrate CRM107 total amount (mg) 0 0 0 0 24.8Microfiltration Filtrate CRM107 total amount (%) 100 100 100 100 100 100Ultrafiltration Retentate CRM107 total amount (%) 85.5 85.5 137.1 61.262.0 N/A Ultrafiltration Filtrate CRM107 total amount (%) 0 0 0 0 30.6Diafiltration Retentate CRM107 total amount (%) 102.9 88.2 97.4 113.624.1 67.5 Diafiltration Filtrate CRM107 total amount (%) 0 0 0 0 23 N/A

TABLE 7 HP-SEC analysis of diafiltration samples from the differentprimary recovery schemes Primary Recovery Scheme Diafiltration Step 1 23 4⁽¹⁾ 5 6 Retentate CRM107 amount (peak area)⁽¹⁾ 96.5 77.6 161.2 88.358.1 642.0 Retentate CRM107 amount 9.0 8.8 17.5 6.6 4.8 22.19 (% totalpeak area) Retentate CRM107 conc. by SDS-PAGE 1.396 1.197 1.271 0.8340.358 3.429 (g/L) ⁽¹⁾100 μL samples were loaded on the column

EXAMPLE 3 Purification of CRM107 with Ion Exchange Chromatography andHydrophobic Interaction Chromatography Background

CRM107 purification protocol has traditionally involved twochromatographic processes. First is HIC which leads to the greatestfold-purification, removing most of the contaminating pigment in theprocess, but has the disadvantage of the greatest drop in yield. Next,after concentration and exchange into the appropriate buffer viadiafiltration, an IEC step is performed to further clean the product;the loss in yield is not as dramatic but neither is the degree ofpurification.

This study reverses these two chromatographies to determine if animprovement could be made in either yield or purity without adverseeffects, the rationale being that not only would the IEC procedure actas a gross scrubbing step for the HIC run but it would also be moreamenable to the increased sample volume anticipated with large scalefermentations of 50 L.

Materials and Methods 1. Preparation of Samples

A crude, frozen preparation of CRM107 consists of 20 L of cell freefermentation broth from a 50 L fermentation concentrated across a 30Khollow-fibre filter to yield 2 L of retentate. This was diafilteredfurther (using a 10K Omega Ultrasette) to equilibrate the pH andconductivity with Buffer C (10 mM Potassium Phosphate, pH 7.6±0.2); theresulting 400 mL retentate was split into 200 mL aliquots for storage at−70° C. Prior to the initial chromatographic step each lot was thawed atapproximately 27° C.; hereafter the procedures differ.

The sample purified according to the traditional protocol is referred toas LOT A while the one subjected to the alternate procedure is labelledLOT B.

(A) The 200 mL of retentate was conditioned for HIC by adding 30 g of(NH₄)₂SO₄ with stirring. This material was then passed through a 0.22μfilter (Media-Kap 25) to remedy its cloudiness prior to loading onto theHIC column.

(B) 500 mL of Buffer C was added to 200 mL of retentate in order toadjust the loading to be identical to that of the IEC step of Lot A (seeSection 3). No filtration was necessary.

2. First Chromatographic Procedure (A) Hydrophobic InteractionChromatography Using Decyl Agarose 6XL.

A column of Decyl Agarose 6XL column volume {V_(c)} ˜250 mL) wasequilibrated with Buffer A (50 mM Potassium Phosphate, 1 M (NH₄)₂SO₄, pH7.0±0.2) prior to loading of the filtrate obtained after the 0.22 μmfiltration. After loading the column was washed with four V_(c) ofBuffer A, followed sequentially by five V_(c) of Buffer B (50 mMPotassium Phosphate, pH 7.0±0.2) at each of 30% B, 50% B, and 100% B.CRM107 was eluted during the 30% B step and its purity was measured as55.1% by HP-SEC. All pools were stored @ 2-8° C. pending furtherprocessing.

(B) Ion Exchange Chromatography Using Pharmacia DEAE Fast Flow.

A Pharmacia DEAE FF column (V_(c)˜220 mL) was equilibrated with Buffer Cprior to loading the 700 mL sample. It was then washed with four V_(c)of Buffer C. Bound CRM107 was eluted with a linear gradient of 0-50%Buffer D (10 mM Potassium Phosphate, 500 mM KCl, pH 7.6±0.2) carried outover ten V_(c) with concurrent collection of 90 mL fractions. It wasthen washed with five V_(c) of 100% D. After HP-SEC analysis, it wasdecided to pool fractions 12-16; the resulting purity was approximately69.8% (calculated from the Peak Area and Area Percent of each fraction).Each pool/fraction was collected and stored @ 2-8° C. until furtherprocessing.

3. Diafiltration & Concentration Using 10K Omega Ultrasette

(A) The 1300 mL pool of CRM107 from the HIC step (the 30% B fraction)was diafiltered using a 10K Omega Ultrasette membrane. Diafiltration wascarried out using Buffer C until the conductivity of the pooled samplefell to ≦5 mS/cm; 3900 mL of diafiltration buffer was required to reacha conductivity of 4.34 mS/cm and a final retentate volume of 700 mL. Thepurity of CRM107 in the retentate was measured as 79.3% by HP-SEC. Thepools were stored @ 2-8° C. until further processing.

(B) The 450 mL pool of CRM107 from the DEAE step (fractions 12-16) wasdiafiltered using a 10K Omega Ultrasette membrane. Diafiltration wascarried out using Buffer A until the conductivity of the pooled samplerose to 130±20 mS/cm; 600 mL of diafiltration buffer was required toreach a conductivity of 116 mS/cm and a final volume of 300 mL. Thepurity of CRM107 in the retentate was measured as 84.2% by HP-SEC. Thepools were stored @ 2-8° C. until further processing. The solutionremained clear throughout this step.

4. Second Chromatographic Procedure (A)—Ion Exchange ChromatographyUsing Pharmacia DEAE Fast Flow.

A Pharmacia DEAE FF column (V_(c) ˜220 mL) was equilibrated with BufferC prior to loading the 700 mL sample. It was then washed with four V_(c)of Buffer C. Bound CRM107 was eluted with a linear gradient of 0-50%Buffer D carried out over ten V with concurrent collection of 90 mLfractions. It was then washed with five V_(c) of 100% D. After HP-SECanalysis, it was decided to pool fractions 15 & 16; the resulting purityis 94.9% (95.6% if calculated from the Peak Area and Area Percent ofeach fraction as per Section 2, LOT B). Each pool/fraction was collectedand stored @ 2-8° C. until further processing.

(B)—Hydrophobic Interaction Chromatography Using Decyl Agarose 6XL

A column of Decyl Agarose 6XL (V_(c) ˜220 mL) was equilibrated withBuffer A prior to loading of the 300 mL sample. After loading the columnwas washed with four V_(c) of Buffer A, followed sequentially by fiveV_(c) of Buffer B at each of 30% B, 50% B, and 100% B. CRM107 was elutedduring the 30% B step and its purity was measured as 76.6% by HP-SEC.All pools were stored @ 2-8° C. pending further processing.

5. Second Diafiltration & Concentration Using 10K Cutoff

(A) Buffer C was used to equilibrate a 150 mL Filtron stirred-cell unitcontaining a 10K Omega membrane, pressurized to 40-45 psi. This was usedto diafilter the 180 mL DEAE pool. 260 mL of Buffer C was used to yield31 mL of retentate with a final purity of 98.0% as monitored by HP-SEC.The HP-SEC profile had no detectable peaks with retention times between10 and 11 minutes.

(B) The 1100 mL HIC pool was first concentrated using the 10K OmegaUltrasette; 670 mL of Buffer C was used to bring the retentate volumedown to 150 mL. This was further concentrated to 34 mL with the prepared150 mL Filtron stirred-cell unit containing a 10K Omega membrane. Thefinal purity was 97.4% as monitored by HP-SEC. The HP-SEC profile showedtwo peaks at 10.2 and 10.6 minutes which account for 1.4% of the AreaPercent.

Results and Discussion

To determine the efficiency of each step, various in-process sampleswere analysed by some or all of the following techniques: HP-SEC,SDS-PAGE, IEF, Protein-dye (Coomassie) absorbance, intrinsic absorbanceat 280 nm, & DNA-dye fluorescence. These data are summarised in Tables 8and 9.

Whilst the procedures do appear to differ in their overall percentrecoveries this is partially an artifact of the excessive backpressureapplied to the Ultrasette diafiltration step performed on Lot A.Consequently, about 60% (160 mg) of the CRM107 fraction from the HICstep was lost to the filtrate, compared to only 8% for Lot B. All elsebeing equal, when this loss is factored in Lot A should have had anoverall recovery of 27% (160 mg), more on par with Lot B. Even with thisirregularity accounted for, the overall yield for Lot A is still only70% of that obtained with Lot B.

TABLE 8 Yield, purity and recovery data for Lots A and B A B Step HP-SECStep HP-SEC Yield Recovery Purity Yield Recovery Purity Step (mg) (%) (%Area) (mg) (%) (% Area) Crude CRM107 600 N/A 22.2 600 N/A 22.2Preparation for 400 67 21.1 600 100  17.8 Chromatography 1stChromatography 270 68 55.1 400 67 69.8 Pool¹ Ultrasette Pool 85  31^(‡)79.3 370 92 84.2 2nd Chromatography 37 44 94.9 130 35 76.6 Pool²  (54)* (57)* Stirred Cell 46 124  98.0 210 163  97.4 (Final Product) OverallRecovery 7%[27%]‡ 35% ^(‡)The Ultrasette was subjected to excessivebackpressure during this step as evidenced by the amount of CRM107 inthe filtrate (156 mg, ~60% of the HIC pool); during B only 30 mg (8% ofthe DEAE pool) was similarly lost. Taking this loss into account, theoverall recovery for Lot A is 27% *These values were calculated usingstirred cell yields

TABLE 9 Results for Purification of CRM107 from Lots #A and B A B CrudeHIC Ultrasette DEAE Stirred Crude DEAE Ultrasette HIC Stirred SampleCRM107 Pool Pool Pool Cell CRM107 Pool Pool Pool Cell CRM107 Notdetermined due to 45 mg Not determined due to 180 mg yield byinterfering species interfering species absorbance at 280 nm CRM107 35mg 160 mg yield by Coomassie dye-binding [DNA] N.D. 800 280 <LOD <LODN.D. <LOD <LOD <LOD <LOD (ng/mL) IEF Indistinguishable from the 0.4 g/LIndistinguishable from the 0.4 g/L CRM107 Standard CRM107 Standard Note:The LOD (Limit of Detection) for the DNA assay is 5 ng/mL

This discrepancy between the two overall recoveries is undoubtedlydirectly attributed to the different processes used to prepare thesamples for the first chromatography step. In Lot A, the additional(NH₄)₂SO₄ and filtration steps led to the loss of 200 mg of the CRM107present in the culture supernatant.

CRM107 produced from Lot B by performing IEC before HIC led to anincrease in the amount recovered from the concentrated broth.

EXAMPLE 4 Expression of CRM107 in Escherichia coli

This was to study the expression of the diphtheria toxin mutant CRM107 adifferent microbial host. DNA was purified from corynebacteriophage βisolated from cultures C. diphtheriae, and a 2 kb fragment containingthe entire gene encoding CRM107 including the promoter and the signalsequence was inserted into the polylinker cloning site of pUC19 andamplified in Escherichia coli. CRM107 was expressed in different E. colistrains and accumulated in the periplasm of the bacteria. The expressionlevel was up to 45 mg recombinant protein per litre medium.

Materials and Methods

Strains and plasmids: Corynebacterium diphtheriae C7(β^(tox-107)) wasreceived from Dr. Richard Youle, NIH, and was used for production ofCRM107. C. diphtheriae C7(−) (ATCC 27010) was used for amplification andtitration of β-phages.

The plasmid pUC19 (Gibco, Paisley, UK) was used as vector for cloning ofthe CRM107 gene and E. coli DH5α (Gibco), E. coli HB2151 (PharmaciaBiotech, Uppsala, Sweden), E. coli TG1 (Pharmacia Biotech) and E. coliRR1ΔM15 (ATCC 35102) were used for expression of CRM107.

Preparation of β-phage: DNA was prepared as described by Greenfield, L.,Bjorn, M. J., Horn, G. Fong, D., Buck, G. A., Collier, R. J. and KaplanD. A. (1983) Proc. Natl Acad Sci USA 80, 6853-6857 with minormodifications. Briefly, β-phages were isolated from the medium of C.diphtheriae C7(β^(tox-107)) and amplified in C. diphtheriae C7(−). Thecells were removed by centrifugation at 2500 g, and the medium wasfurther clarified by centrifugation at 1500 g. Polyethylene glycol 8000and NaCl were added to concentration of 6% and 0.5 M, respectively, tothe phage-containing supernatant. The phages were precipitated byincubation on ice for 2 hours and recovered by centrifugation at 15000 gfor 20 min.

Titration of phages to determine the number of plaque forming units(pfu) was performed in C. diphtheriae C7(−) as described in Laird, W andGroman, N. (1976) J. Virol 19, 208-219.

The phages (10¹¹ pfu) suspended in 100 mM Tris-HCl, pH 7.5, 100 mM NaCl,25 mM EDTA were incubated with 1 mg/ml pronase (Sigma, St. Louis, Mo.)for 2 hours at 37° C. and then extracted with an equal volume ofphenol:chloroform:isoamylalcohol (49.5:49.5:1). The aqueous phase wasprecipitated with cold ethanol after addition of Na-acetate to aconcentration of 0.3 M. The pellet was washed once with 70% ethanol anddissolved in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.

Cloning of CRM107 into pUC19. Purified β-DNA was cleaved with EcoRI andXbal and separated in 1% agarose gel. A band of 2 kb was excised fromthe gels and purified using a QIAEXII Agarose Gel Extraction Kit (QiagenGmbH, Hilden, Germany). The isolated fragment was ligated into thepolylinker cloning site of pUC19. Restriction enzymes (New EnglandBiolabs, Beverly, Mass.) and T4 ligase (Pharmacia Biotech) were usedaccording to the suppliers recommendations. The resulting plasmid,pEtox, was amplified in E. coli TG1.

Expression of CRM107 in E. coli: The plasmid pEtox was transformed todifferent E. coli strains by electroporation. The bacteria are grown at37° C. in accordance with the invention. The cells are harvested bycentrifugation, and whole cell extracts made by disintegration of thecells in a French Press. Fractionation of the cells was performed byfirst isolating the periplasma proteins from freshly grown cells byosmotic shock. Nossal, N. G. and Heppel, L. A. (1966) J. Biol Chem 241,3055-3062. The residual was disintegrated in a French Press, and themembrane fraction separated from the cytoplasma by centrifugation.

EXAMPLE 5 Conjugation of Transferrin with CRM107

CRM107 was conjugated with transferrin according to published methods,including the method described in U.S. Pat. No. 5,728,383.

Substitution of CRM107:

150 mg of CRM107 (concentration of approximately 10 mg/ml) was mixedwith 299 μl of MBS solution (10 mg/ml) and incubated at room temperaturefor 30 minutes. The amount of MBS added was a 3.5 times molar excess ofCRM107. Upon completion of the incubation, the reaction was quenched byplacing the mixture at 5° C., followed by desalting on a Sephadex G25column.

Substitution of Transferrin:

350 mg of transferrin (concentration approximately 20 mg/ml) was mixedwith 840 μl 2-IT (5 mg/ml) and incubated at 37° C. for 30 minutes. Theamount of 2-IT added was 7.7 times molar excess of transferrin. Uponcompletion of the incubation the reaction was quenched by placing themixture at 5° C. followed by desalting on a Sephadex G25 column.

Conjugation of Substituted Transferrin and CRM107:

The MBS substituted CRM107 (141 mg) and 2-IT substituted transferrin(293 mg) in the ratio of 1:2 were mixed together and incubated at 5° C.for 18 hours. Upon completion of the conjugation the conjugated waspurified by ion exchange chromatography using Q Sepharose Fast Flowfollowed by gel filtration on Superdex 200 column.

Anion Exchange Chromatography of Conjugate:

Approximately 440 mg of conjugate was loaded onto 100 ml Q SepharoseFast Flow column, equilibrated with sodium phosphate (0.05M) buffer atpH 7.6. The conjugate was eluted with 300 ml (3 column volumes) ofelution buffer consisting of 70% equilibration buffer and 30% Buffer B(0.1M sodium phosphate, 1.5M sodium chloride pH 7.4). The conjugate peakwas eluted between 60 ml and 150 ml of elution buffer.

Concentration of Conjugate:

Fractions from the Q Sepharose Fast Flow column which contained theconjugate were pooled and concentrated to a concentration greater than10 mg/ml and loaded onto a Superdex 200 molecular sieve column forfurther purification. Equilibration and elution buffer for the Superdex200 was 0.01M sodium phosphate pH 7.4 containing 0.15M sodium chloride.Purified conjugate was stored at −70° C.

1-22. (canceled)
 23. Diphtheria toxin having a purity of at least 98%.24. Diphtheria toxin when produced by the method of fermenting amicroorganism capable of producing diphtheria toxin using glucose as acarbon source and adding glucose to a growing culture whereby theaddition of glucose maintains a microorganism growth effective tosupport diphtheria toxin production.
 25. A method for preparing adiphtheria toxin conjugate comprising conjugating a diphtheria toxinwith one of a cell specific binding and targeting moiety, wherein saiddiphtheria toxin is produced by the steps of fermenting a microorganismcapable of producing diphtheria toxin using glucose as a carbon sourceand adding glucose to a growing culture whereby the addition of glucosemaintains a microorganism growth effective to support diphtheria toxinproduction.
 26. The method as claimed in claim 25 wherein the cellspecific binding or targeting moiety is transferrin.
 27. The method oftreatment of CNS neoplasm comprising administering to a subject adiphtheria toxin conjugate as produced by the method of claim
 25. 28.The use of a diphtheria toxin conjugate produced by the method of claim25 in the manufacture of a medicament for use in the treatment of CNSneoplasm.